mTOR Kinase Activity Assay

The mTOR kinase activities of all the compounds were determined using LANCE^® ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assay (Invitrogen, Carlsbad, CA, USA) [20 (link)] following the manufacturer’s instructions, with compound GDC-0941 as positive controls. Briefly, mTOR enzyme (0.1 μg/mL, Invitrogen), ATP (3 μM), GFP-4EBP1 Peptide (0.4 μM) and test compounds were diluted in kinase buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 3 mM MnCl₂, 10 mM MgCl₂, 2 mM DTT and 0.01% Tween-20). The reactions were performed in black 384-well proxiplates (Corning) at room temperature for 1 h and stopped by adding EDTA to 10 mM. Tb-antiphospho-4EBP1 (Thr37/46) Antibody (PerkinElmer, Fremont, CA, USA) was then added to each well to a final concentration of 2 nM, and the mixture was incubated at room temperature for 30 min. The intensity of the light emission was measured with Spectramax 190 reader (Molecular Devices, Valley, CA, USA) in TR-FRET mode (excitation at 320 nm and emission at 665 nm). All of the compounds were tested two times, and the results expressed as IC₅₀ (inhibitory concentration 50%) were the averages of two determinations.